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First published online 9 March 2004
doi: 10.1242/jcs.01018

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Role of tau phosphorylation by glycogen synthase kinase-3ß in the regulation of organelle transport

Yoshitaka Tatebayashi^*, Niloufar Haque, Yunn-Chyn Tung, Khalid Iqbal and Inge Grundke-Iqbal

New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York, NY 10314, USA

View larger version (77K): [in a new window]   Fig. 1. Effect of FGF-2 and NGF on the differentiation of PC12 cells with respect to morphology and levels of MAP1B and tau. (A) Representative cell morphologies during differentiation (4 and 10 days) of PC12 cells in the presence of NGF or FGF-2. Note that PC12 cells after differentiation for 10 days with NGF have a larger number of thicker neurites than those with FGF-2. Bar, 50 µm. (B) Western blot showing MAP1B in PC12 cells differentiated with NGF or FGF-2 from 0 to 10 days. (C) Quantitative analysis of total tau and MAP1B levels in PC12 cells differentiated with NGF or FGF-2. Immunoreactivities (IR) of tau or MAP1B were converted based on the value (PSL) of these proteins from undifferentiated cells as 100%. Tau was analyzed on 125I western blots with Tau-1 after 8 hours dephosphorylation on the membrane with alkaline phosphatase. The increase of tau expression after 7 days of differentiation was confirmed with the phospho-independent tau antibody R134d and was 3.5 and 4 times higher (data not shown). MAP1B was analyzed with mAb 3G5.

View larger version (50K): [in a new window]   Fig. 2. Differentiation of PC12 cells with NGF or FGF-2 upregulates the expression of tau and GSK-3ß and phosphorylation of tau at the Tau-1 site. (A) 125I western blots of tau and GSK-3ß in PC12 cells differentiated with NGF or FGF-2; 12 µg of total cell lysate was applied per lane for SDS-PAGE. For immunostaining of tau with Tau-1 antibody, the blots were untreated (Tau-1) or pretreated with alkaline phosphatase (Tau-1 dp). Arrowhead indicates the position of high molecular mass tau; bars on the right indicate the positions of 70 kDa and 50 kDa molecular mass markers (from top). For GSK-3ß immunostaining, the blots were developed with antibody 127d to total GSK-3ß and with antibody Y216 to GSK-3ß phosphorylated at Tyr 216. (B) NGF and FGF-2 upregulate GSK-3ß activity in differentiated PC12 cells. GSK-3ß activities were measured by using 127d-immunoprecipitates of the extracts of cells cultured in NGF or FGF-2 for the indicated time periods. The activities (%) were converted based on the value of unstimulated cells as 100% (mean±s.e.m.). (C) NGF and FGF-2 increase tau phosphorylation at the Tau-1 site. The percentage of phosphorylation at the Tau-1 site was determined as described in Materials and Methods (mean±s.e.m.). (D) Lithium and valproate (VA) inhibit tau phosphorylation at the Tau-1 site in differentiated PC12 cells. PC12 cells differentiated by NGF for 4 days were further incubated without (control, C) or with LiCl (20 mM, 3 hours, LiCl), butyrolactone 1 (10 µM, 6 hours, Bu1), PD98059 (50 µg/ml, 1 hour, PD) or VA (0.6 mM, 3 hours, VA), lysed and analyzed by western blots developed with Tau-1 (20 µg of cell lysate per lane). Not shown in this figure, these treatments did not affect total tau levels significantly as determined by immunolabeling with Tau-1 after dephosphorylation. Only the GSK-3 inhibitors, lithium and VA increased Tau-1 staining. (E) GSK-3 inhibitors alsterpaullone and LiCl inhibit the GSK-3 enzyme activity in PC12 cells. PC12 cells were treated for 1.5 hours with medium (C), 10 µM butyrolactone I (Bu), 20 µM alsterpaullone (Alst) or 20 mM lithium chloride (LiCl). Cells were lysed and the GSK-3 enzyme activity determined. *P<0.001.

View larger version (45K): [in a new window]   Fig. 3. Site-specific effect of LiCl and Bu1 on tau phosphorylation in differentiated PC12 cells. PC12 cells were differentiated with FGF-2 for 7 days and then treated with medium alone (C), or medium containing 20 mM LiCl (L) or 10 µM Bu1 (B). (A) The cell lysates (20 µg/lane) were analyzed by 125I western blotting with phospho-independent antibody R134d to tau and antibodies to different phosphorylation sites on tau (indicated at the bottom of each individual panel). Bars on the left indicate the positions of 70 kDa and 50 kDa molecular mass markers. (B) Quantitative evaluation of the effect of the inhibition of GSK-3 with LiCl (white bars) and of cdk5 with Bu1 (hatched bars) on the phosphorylation of tau at different sites. The values represent the degrees of inhibition of site specific phosphorylation calculated after normalization of each individual value with the corresponding values for total tau (R134d). Values are means of three to nine individual western blots. Bars represent s.e.m.; *P<0.05, **P<0.01, ***P<0.001, Student's t-test. (C,a) Western blot analysis of tau bound (B) or unbound (U) to microtubules in differentiated PC12 cells (NGF, for 4 days) pretreated with (Li+) or without (Li–) 20 mM LiCl for 3 hours. Blots were untreated (Tau-1) or treated with alkaline phosphatase (Tau-1 dp) and stained with Tau-1 or directly stained with DM1A for -tubulin (data not shown). Lines on the right side of the western blots indicate the positions of the 70 kDa and 50 kDa molecular mass markers. (b) Quantitative analysis of the degree of phosphorylation of tau at the Tau-1 site from a in the bound fraction. Lithium (Li+) markedly decreased tau phosphorylation at the Tau-1 site. (c) Quantitative analysis of the ratio of bound tau to unbound tau (B/U) from a (mean±s.e.m.). Lithium (Li+) did not affect tau's ability to bind to microtubules (compare Li- with Li+).

View larger version (59K): [in a new window]   Fig. 4. Inhibition of GSK-3ß but not of cdk5 induces reversible mitochondrial clustering in differentiated PC12 cells in association with dephosphorylation of tau at the Tau-1 epitope. (A) PC12 cells differentiated with FGF-2 for 5 days were treated with medium alone (a-c,m-o), or medium with GSK-3 inhibitors LiCl (g-i), VA (j-l) and alsterpaullone (images not shown) or the cdk5 inhibitor butyrolactone 1 (d-f) for 1.5 hours. The cells were then fixed and immunostained with Tau-1 (a,d,g,j,m) and the distribution of mitochondria was visualized with Mito Tracker Red® which had been added after 1 hour of incubation with kinase inhibitors (b,e,h,k,n). The size of the cells and their processes can be seen by phase contrast microscopy (c,f,i,l,o). Inhibition of GSK-3 induced mitochondrial clustering associated with increased staining at the Tau-1 epitope (dephosphorylation) as well as loss of mitochondria from the neurites (g-i,j-l) and the periphery of the cell body (compare l with o). Inhibition of cdk5 (d-f) resulted in flattening of cells and processes but neither mitochondrial movement nor Tau-1 phosphorylation were affected. Images in a, b, d, e, g, h are maximum projections of four confocal captured sections (j,k,m,n) are fluorescence micrographs. Arrowheads in j-o indicate the position of the cell membrane as seen in the corresponding phase contrast images (l,o). Scale bars: 10 µm. (B) Partial reversal of mitochondrial clustering. PC12 cells differentiated with NGF for 4 days were treated with 20 mM LiCl for 1 hour (a). Cells were then washed and incubated in medium without LiCl for another 1 hour (b). The distribution of mitochondria was visualized with Rhodamine 123 through FITC filter using fluorescence microscopy. More intense signals were observed in the periphery of the cells and the neurites after washing out lithium (b). Since it is unlikely that mitochondria sequester additional dye during the wash-out period of LiCl in the conditions used, the increased signals in the periphery and the neurites in b most probably reflect mitochondria transported anterogradely during the wash-out period. Arrowhead indicates restoration of mitochondrial distribution following washing out of LiCl; arrow indicates newly appeared neurites; identical color saturation in a and b. Scale bars: 10 µm.

View larger version (76K): [in a new window]   Fig. 5. Mitochondrial clustering in tau transfected CHO cells in which tau is not phosphorylated at the Tau-1 site. (a-f) CHO cells transfected with tau were triple stained with Mito Tracker Red® (mitochondria) and antibodies R134d to total tau (green) and Tau-1 to unphosphorylated tau (blue). Arrows indicate the cells with mitochondrial clustering, arrowheads indicate the cells with normal mitochondrial distribution. Both arrows and arrowheads show the position of the cell membrane seen by phase microscopy (not shown). In cells with unphosphorylated tau (blue) clustering of mitochondria was observed (a,d; arrows). In contrast, the distribution of mitochondria was almost normal in cells containing phosphorylated tau (a,d; arrowheads). Asterisks indicate non-tau-expressing cells (e,f) with normal distribution of mitochondria (d). Tau-transfected CHO cell (g,h; left) with mitochondria clustered around the nucleus, double stained (g) for tau (R134d, green) and mitochondria (Mito Tracker Red®). The cell on the right in g, h is not transfected. Orange in g indicates overlapping areas of tau and mitochondrial stains. (h) Phase micrograph of g showing that the periphery of the transfected cell (g,h; left) is practically free of organelles. Scale bars: 10 µm.

View larger version (104K): [in a new window]   Fig. 6. Lithium inhibits the initial neurite outgrowth of PC12 cells and neural progenitor cells derived from adult rat hippocampus. (A) Excess myo-inositol administration failed to rescue the inhibition of neurite outgrowth of PC12 cells by LiCl. PC12 cells induced to differentiate by NGF in the presence of excess myo-inositol (20 mM) were left untreated (NGF + myo) or simultaneously treated with lithium (NGF + myo + LiCl) for 3 days. Scale bar: 25 µm. (B) AHPs expanded by FGF-2 (10 ng/ml) were replated on culture slides with 40 ng/ml FGF-2 with or without 20 mM LiCl for 1 day. (a,b) Cell morphologies of AHPs without (a) or with (b) lithium. Scale bar: 10 µm. (c) Quantitative data showing the percentage of neurite-bearing cells. PC12 cells possessing one or more neurites of a length more than 1.5-fold the diameter of the cell body were scored as positive. Lithium almost completely blocked initial neurite outgrowth of AHPs (mean±s.e.m.). P<0.001.

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